Cancer that originates in squamous epithelia can develop in a large number of organs or tissues. The appearance of (pre)cancerous tissue can show very large differences depending on the body site, as can the difficulties and consequences related to excision of malignant tissue. This is especially relevant for cervical cancer which is the leading cause of cancer death in women in many developing countries and where the early detection and precise excision of malignant tissue are determining for the patient's ability to bear children afterwards.
A physician doing an excision procedure of a cervical cancer or cervical intraepithelial neoplasia (CIN) site faces two conflicting objectives: On one hand he wants to excise as little tissue as possible (especially if the patient wants to preserve her ability to bear children) on the other hand he has to excise all (pre-) cancerous tissue (with a safety margin) to avoid a recurrence. In practice however, the physician cannot directly see the lesion. Instead he/she has to rely on colposcopy images and/or on staining or marking techniques. The most commonly used marking technique for cervical cancer is acetowhitening. Both colposcopy images and acetowhitening are notoriously hard to interpret with large observer-to-observer variations. In addition, the acetowhite staining is transient, i.e. the white pattern will change while the excision takes place. FIGS. 1A-C schematically depicts an excision procedure of an ectocervical lesion; the black circle indicates the location of the cervical os. The exact extent of the lesion is unclear (1A), so the physician excises what he thinks is the approximate extent of the lesion (1B). After the excision (1C) the acetowhite is no longer visible on the remaining or the excised tissue, so there is no direct feedback to the physician on whether he/she was successful. The patient has to wait for the histopathology report.
Also these methods provide only information about tissue which can be assessed visually. For e.g. cervical cancer this will be the Ectocervix (which is all that is visible with the aid of a colposcope). How far a lesion extends into the endocervix is not known. Even a very experienced physician will be confident only of the approximate lateral extension of the lesion he/she is trying to remove. How deep it has to be removed remains a guess work. Most physicians will have to try to do a precise excision while actually being virtually blind.
In acetowhitening, 3-5% acetic acid produces a coagulation reaction with cells which is proportional to the content of nuclear protein in the cell. This reaction produces a noticeable effect compared with the normal pinkish color of the surrounding epithelium that is commonly visible to the naked eye.
With low-grade CIN, the acetic acid must penetrate into the lower one-third of the epithelium (where most of the abnormal cells with high nuclear density are located). Hence, the appearance of the whiteness is delayed and less intense due to the smaller amount of nuclear protein compared to areas with high-grade CIN or preclinical invasive cancer. Areas of high-grade CIN and invasive cancer turn densely white and opaque immediately after application of acetic acid, due to their higher concentration of abnormal nuclear protein and the presence of large numbers of dysplastic cells in the superficial layers of the epithelium.
The acetowhite appearance is not unique to CIN and early cancer. It is also seen in other situations when increased nuclear protein is present: for example in immature squamous metaplasia, congenital transformation zone, in healing and regenerating epithelium (associated with inflammation), leukoplakia (hyperkeratosis) and condyloma. While the acetowhite epithelium associated with CIN and preclinical early invasive cancer is more dense, thick and opaque with well demarcated margins from the surrounding normal epithelium, the acetowhitening associated with immature squamous metaplasia and regenerating epithelium is less pale, thin, often translucent, and patchily distributed without well-defined margins. Acetowhitening due to inflammation and healing is usually distributed widely in the cervix, not restricted to the transformation zone. The acetowhite changes associated with immature metaplasia and inflammatory changes quickly disappear, usually within 30-60 seconds.
Acetowhitening associated with CIN and invasive cancer quickly appears and persists for more than one minute. The acetic acid effect reverses much more slowly in high-grade CIN lesions and in early pre-clinical invasive cancer than in low-grade lesions, immature metaplasia and sub-clinical HPV changes. It may last for 2-4 minutes in the case of high-grade lesions and invasive cancer.
In short, acetowhitening correlates with the content of nuclear protein in the cells, and thus marks all tissue states having a substantially increased level of nuclear protein. The appearance of the acetowhite (density, intensity, opaqueness) depends on the type and exact location of the nuclear protein as well as on the time after application of the acetic acid (delay and decay of whitening). For suspected cervical cancer, acetowhitened tissue regions are notoriously hard to interpret and only the Ectocervix is marked up. How far a lesion extends into the endocervix cannot be determined.
In conclusion, in present methods applying acetowhitening during excising procedures, acetowhitening simultaneously detects and marks potential (pre)cancerous tissue. The application of acetic acid first marks up tissue with elevated nuclear protein content, all of which is potentially (pre)cancerous tissue, where after the physician evaluates which of the marked up tissue is actually (pre)cancerous based on his her level of skill and experience. Acetowhitening thereby involves the main disadvantages that it is very difficult to apply any objective threshold in the classification of the malignancy of the tissue and that the marking is not permanent leading to practical difficulties when performing excision procedures. Additionally, the endocervix is difficult to mark up, and especially it will be difficult to detect marked tissue in the endocervix.
The inventor of the present invention has appreciated that an improved methodology and apparatus for detecting and marking cancerous tissue is of benefit, and has in consequence devised the present invention.